Thick filament regulation of myocardial contraction

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita."August 2006"Thesis (Ph. D.) University of Missouri-Columbia 2006.The ability of the heart to function as a pump is governed by mechanisms intrinsic to individual cardiac myocytes. The experiments in this dissertation were designed to examine the effects of sarcomere length and thick filament protein isoform expression on the contractile properties of single skinned cardiac myocytes. Myosin binding protein-C ablation (MyBP-C-\-) increases the rate of force development, loaded shortening velocity, and power output in mouse skinned cardiac myocytes, implying that MyBP-C regulates myocardial contractility by limiting crossbridge cycling. We also examined the effects of SL on mechanical properties in rat skinned cardiac myocytes containing either [alpha]-MyHC or [beta]-MyHC. Peak absolute and normalized loaded shortening velocity and power output was decreased at short SL in both [alpha]-MyHC and [beta]-MyHC myocytes. Matching myocyte force between long and short SL, however, sped loaded shortening velocity and increased power output in [alpha]-MyHC myocytes to values greater than at long SL, but this did not occur in [beta]-MyHC. Matching myocyte width between long and short SL sped loaded shortening velocity and increased power output to values greater than at long SL in both [alpha]-MyHC and [beta]-MyHC myocytes. It is concluded that there is an increase in crossbridge cycling at short SL as compared to long SL, but increased lattice spacing at short SL decreases actomyosin interactions. The data are presented in terms of a model whereby shortening SL induces a conformational change in MyBP-C that removes its constraint on the myosin heads, allowing them to cycle faster.Includes bibliographical reference

What role does phosphorylation of cardiac troponin I play in elevating cardiac contractility following adrenergic stimulation?

Abstract only availableMyocardial performance is enhanced when adrenergic receptors are stimulated by catecholamines. The enhanced performance is mediated by cAMP dependent protein kinase A (PKA), which phosphorylates several proteins within the cardiac myocyte including cardiac troponin I (cTnI) and myosin binding protein-C (MyBP-C). Phosphorylation of these two proteins by PKA appears to mediate inotropic effects of adrenergic stimulation by directly modulating the rate of cross-bridge cycling. For instance, phosphorylation of cTnI and MyBP-C by PKA increased the power generating capacity of single permeabilized cardiac myocyte preparations (Herron, Korte, McDonald Circ Res 89;1184-1190:2001). It is unknown whether phosphorylation of cTnI or MyBP-C alone or phosphorylation of both proteins is necessary to increase power. The purpose of this study was to develop a methodology to test the relative importance of the two PKA phosphorylation sites on cTnI (serine 23 and 24) in mediating the PKA induced increase in myocyte power output. For these experiments, serines 23 and 24 of rat cTnI cDNA were mutated to alanines using site directed mutagenesis. Next, mutated cTnI (cTnI23/24) was expressed in E. coli bacteria and purified using ion exchange chromatography. Mutated cTnI (cTnI23/24) was then complexed with purified cTnT and cTnI and this whole troponin (cTn) complex was exchanged for endogenous cTn in permeabilized cardiac myocytes overnight using a cTn exchange buffer (20 mM Imidazole, 200 mM KCl, 5 mM EGTA, 5 mM MgCl2, 1 mM DTT). The extent of cTn exchange was assessed by quantifying the amount of PKA-induced phosphate incorporation. We observed only a partial reduction in PKA-induced phosphate incorporation following exhange of cTnI23/24, implicating only a partial Tn exchange using these conditions. We are currently seeking to increase the extent of cTnI23/24 exchange after which individual myocyte preparations will be mounted between a force transducer and position motor and myocyte power output generating capacity will be measured before and after PKA induced phosphorylation of myofilament proteins. These experiments will directly assess whether phosphorylation of cTnI by PKA is necessary to elevate myocyte power output.Life Sciences Undergraduate Research Opportunity Progra

Power Output Is Increased After Phosphorylation of Myofibrillar Proteins in Rat Skinned Cardiac Myocytes

This work was supported by American Heart Association Beginning Grant-in-Aid 9914291 and NIH Grant HL57852.The publisher's version may be found at http://circres.ahajournals.org/cgi/content/full/89/12/1184ß-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca2+ sensitivity of force. PKA also significantly increased both maximal force (25.4±8.3 versus 31.6±11.3 µN [P<0.001, n=12]) and peak absolute power output (2.48±1.33 versus 3.38±1.52 µW/mg [P<0.05, n=5]) during maximal Ca2+ activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased {approx}20% during maximal Ca2+ activations (n=5) and {approx}33% during half-maximal Ca2+ activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output-generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after ß-adrenergic stimulation